Nitric oxide (NO) is involved in numerous physiological functions, including vasodilatation, neurotransmission, and cytotoxic actions of the immune system. NO is produced physiologically by the enzyme nitric oxide synthase (NOS) from the amino acid L-arginine. There are three isoforms of NOS in mammals (endothelial, neuronal, and inducible), each one evolving specific mechanisms and chemistries to suit their unique physiological roles. Determining the catalytic and regulatory mechanisms of NOS isoforms at the molecular level is critical for understanding how NO is produced and managed physiologically, and for designing therapeutic agents that selectively target each NOS isoform. Our long-term goal is to define the molecular mechanisms behind the regulation and production of NO by NOS, providing a better understanding of how these processes are controlled and regulated. Our objective is to answer the following questions: 1) How do conformational changes induced by the binding of cofactors and substrate influence the reactivity of the heme active site? 2) What are the fast catalytic intermediates during the mechanism of NO production? Our central hypothesis is that the binding of cofactors to NOS induces conformational changes that directly affect the active site, with the rationale that understanding the mechanisms for how heme reactivity is modulated by cofactor and substrate binding is crucial for understanding how NO is produced and managed endogenously. In this proposal, we aim to: 1) Determine the mechanism of how the binding of calmodulin alters the reactivity of the heme active site in neuronal NOS. 2) Identify and characterize fast intermediates during each step of the catalytic cycle. These aims will be accomplished using multi-channel (200-800 nm) laser-based nanosecond time-resolved spectroscopy with flow-flash mixing combined with focused mutagenesis to determine how cofactor and substrate binding influences heme reactivity. Since NOS enzymes play diverse roles in human health and disease pathogenesis, we desire to determine how the protein matrix regulates activity and to clarify the mechanism of catalysis. The molecular mechanism of NOS regulation and the clearer description of the mechanism of catalysis that will result from this work will advance the understanding of the role that NOS plays in disease and health. Understanding how NOS is regulated and clarifying its catalytic mechanism are crucial both for designing therapies that control NO synthesis and for understanding how compromised NO physiology leads to deleterious health effects.